Project Summary This proposal seeks to understand the impact of metabolism on cancer aggressiveness, and how metabolic vulnerabilities can be targeted to improve patient outcome. Although targeted therapies have a great focus in the cancer research community, they have failed to generate durable responses, because of the emergence of resistance and the evolution of cancer. Metabolism is exquisitely sensitive to perturbations in the microenvironment, and this is currently an under-investigated area in cancer research. Successfully targeting metabolism has the potential to benefit patients across multiple cancer types, and genotypes, which until now has been a challenge. Cancer preferentially consumes glucose even in the presence of adequate oxygen (aerobic glycolysis), which results in the lactic acid production that decreases extracellular pH. In this Ph.D. project, it is hypothesized an alternative explanation for aerobic glycolysis, also known as the Warburg Effect (W.E), is that the enhanced uptake of glucose is due to the expression of acid exporting membrane transporters. Carbonic anhydrase IX (CA-IX) is one such acid producing protein, which we hypothesize leads to an intracellular proton deficit, driving the fermentation of glucose to replenish the deficit. CA-IX is a clinically relevant protein upregulated in numerous cancers, including breast and ovarian. CA-IX has an exofacial active site that reversibly hydrates CO2 into HCO3- and H+, and we term it a pseudohypoxic protein, as although regulated by hypoxia it is often expressed under normoxic conditions. In Aim 1.1 (prior studies), we have shown that CA-IX, or PMA1(yeast proton ATPase), over-expression in a lowly aggressive, non-metastatic breast cancer cell line (MCF-7) increases the glycolytic rate, glucose uptake, lactate production and increases lung metastasis in vivo. We also developed a metabolic profiling tool to compare 2D and 3D metabolism in the Seahorse Extracellular Flux Analyzer to aid us in our metabolic studies. Finally, preliminary pHi studies show our CA-IX clones have a higher intracellular pH compared to parental MCF-7. In Aim 1.2, (proposed studies), we will take more robust measurements of intracellular pH in both CA-IX and PMA-1 clones at various extracellular pH. We will also measure CA-IX enzymatic activity in the presence and absence of a CA-IX inhibitor from Philogen. We will repeat our in vivo tail vein experimental metastasis studies in the presence and absence of sodium bicarbonate (buffer therapy), and the CA-IX Philogen inhibitor, to see if it reduces metastasis. Finally, in Aim 2, the post-doc will focus on understanding the metabolic phenotype of small cell lung cancer (SCLC). The work will be translationally focused to aid in the treatment of patients. SCLC has a very poor prognostic outcome and, currently, its metabolic vulnerabilities are untapped. Using 13C-labelled metabolite studies in vivo and through patient needle biopsies, we hope to elucidate those metabolic pathways aiding in aggressiveness of the disease. Overall, the focus of my career is to understand the impact of cancer cell metabolism on patient outcome and therapy resistance to clinically benefit patients.